Antibody compositions for preparing enriched dendritic cell preparations

ABSTRACT

The present invention relates to antibody composition that are useful in preparing enriched cell preparations such as human hematopoietic progenitor cells and stem cells and non-hematopoietic tumor cells. The invention also relates to kits for carrying out the processes and to the cell preparations prepared by the processes.

This application is a divisional of U.S. patent application Ser. No.09/088,227 filed on Jun. 1, 1998, now U.S. Pat. No 6,117,985, which is acontinuation-in-part of U.S. patent application Ser. No. 08/566,295,filed Dec. 1, 1995, now abandoned, which is a continuation-in-part ofU.S. patent application Ser. No. 08/491,175, filed Jun. 16, 1995, nowU.S. Pat. No. 5,877,299 all of which are incorporated herein byreference in their entirety.

FIELD OF THE INVENTION

The present invention relates to novel antibody compositions, andprocesses and kits for preparing enriched cell preparations, such ascell preparations enriched in human hematopoietic progenitor cells orstem cells or non-hematopoietic tumor cells.

BACKGROUND OF THE INVENTION

Blood cells have a relatively short life span and need to be replenishedthroughout life. In adults, blood cell formation or hematopoiesis takesplace in the bone marrow, but blood-forming stem cells can also be foundin peripheral blood. Hematopoietic cells represent a hierarchy ofproliferating and differentiating cells. The most abundant are thedifferentiating or lineage committed cells. These cells have limited orno proliferative capacity and represent specialized end cells that arefound in blood, and their immediate precursors.

The immediate precursors of the differentiating cells are the progenitorcells. Most of these cells are restricted to differentiate along asingle lineage but they may have quite extensive proliferative capacity.Progenitor cells appear morphologically as blast cells, and theytypically do not have specific features of the hematopoietic lineage towhich they are committed.

Progenitor cells are derived from stem cells. Stem cells have beenhistorically defined as cells capable of long term hematopoieticrepopulation. This implies their ability to self-renew as well as togenerate daughter cells of any of the hematopoietic lineages. Thepresence of stem and progenitor cells may be detected by their abilityto produce colony-forming cells in culture and repopulate xenogeneichosts such fetal sheep (Zanjani et al., 1994 J. Clin. Invest. Vol. 89,p. 1178-1188) and immuno-deficient mice (Dick et al., 1991 ImmunologicalReviews, Vol. 124:25-43). They may also be detected by screening for theCD34 antigen which is a positive marker for early hematopoietic cellsincluding colony forming cells and stem cells. At present, the long termculture initiating cell (LTCIC) assay appears to be the best way todetect stem cells, or at least the most primitive progenitor cells,using tissue culture methodologies.

There is a continued interest in developing stem cell purificationtechniques. Pure populations of stem cells will facilitate studies ofhematopoiesis. Transplantation of hematopoietic cells from peripheralblood and/or bone marrow is also increasingly used in combination withhigh-dose chemo- and/or radiotherapy for the treatment of a variety ofdisorders including malignant, nonmalignant and genetic disorders. Veryfew cells in such transplants are capable of long-term hematopoieticreconstitution, and thus there is a strong stimulus to developtechniques for purification of hematopoietic stem cells. Furthermore,serious complications and indeed the success of a transplant procedureis to a large degree dependent on the effectiveness of the proceduresthat are used for the removal of cells in the transplant that pose arisk to the transplant recipient. Such cells include T lymphocytes thatare responsible for graft versus host disease (GVHD) in allogenicgrafts, and tumor cells in autologous transplants that may causerecurrence of the malignant growth. It is also important to debulk thegraft by removing unnecessary cells and thus reducing the volume ofcyropreservant to be infused.

Hematopoietic cells have been separated on the basis of physicalcharacteristics such as density and on the basis of susceptibility tocertain pharmacological agents which kill cycling cells. The advent ofmonoclonal antibodies against cell surface antigens has greatly expandedthe potential to distinguish and separate distinct cell types. There aretwo basic approaches to separating cell populations from bone marrow andperipheral blood using monoclonal antibodies. They differ in whether itis the desired or undesired cells which are distinguished/labeled withthe antibody(s).

In positive selection techniques the desired cells are labeled withantibodies and removed from the remaining unlabeled/unwanted cells. Innegative selection, the unwanted cells are labeled and removed.Antibody/complement treatment and the use of immunotoxins are negativeselection techniques, but FACS sorting and most batch wiseimmunoadsorption techniques can be adapted to both positive and negativeselection. In immunoadsorption techniques cells are selected withmonoclonal antibodies and preferentially bound to a surface which can beremoved from the remainder of the cells e,g. column of beads, flasks,magnetic particles. Immunoadsorption techniques have won favourclinically and in research because they maintain the high specificity oftargeting cells with monoclonal antibodies, but unlike FACSorting, theycan be scaled up to deal directly with the large numbers of cells in aclinical harvest and they avoid the dangers of using cytotoxic reagentssuch as immunotoxins, and complement.

Current positive selection techniques for the purification ofhematopoietic stem cells target and isolate cells which express CD34(approximately 1-2% of normal bone marrow) (Civin, C. l., Trischmann, T.M., Fackler, M. J., Bernstein, I. D., Buhring, H. J., Campos, L. et al.1989 Report on the CD34 cluster workshop, In: Leucocyte typing IV, WhiteCell Differentiation Antigens. Knapp, W., Dorken, B., Gilks, W. R.,Reiber, E P., Schmidt, R. E., Stein, H., and Kr. von den Borne, AE.GEds., Oxford University Press. Oxford, pp. 818). Thus, the potentialenrichment of hematopoietic stem cells using this marker alone isapproximately 50 fold. Available techniques typically recover 30-70% ofthe CD34+cells in the start suspension and produce an enrichedsuspension which is 50-90% CD34⁺ (Firat et al., 1988, Bone MarrowTransplantation, Vol. 21:933-938; deWynter, E. A. et al., 1975, StemCells, Vol. 13:524-532; Shpall, E. J., et al. 1994, J. of ClinicalOncology 12:28-36; Thomas, T. E., 1994, Cancer Research, Therapy andControl 4(2): 119-128). The positive selection procedures suffer frommany disadvantages including the presence of materials such asantibodies and/or magnetic beads on the CD34⁺ cells, and damage to thecells resulting from the removal of these materials. Also to beconsidered is the recent evidence that some long term repopulating cellsare CD34⁻ (negative) (Zanjani et al., 1998, Exp. Hematol., Vol.26:353-360) and methods that isolate CD34⁺ will not capture these cells.

Negative selection has been used to remove minor populations of cellsfrom clinical grafts. These cells are either T-cells or tumor cells thatpose a risk to the transplant recipient. The efficiency of these purgesvaries with the technique and depends on the type and number ofantibodies used. Typically, the end product is very similar to the startsuspension, missing only the tumor cells or T-cells.

Transplants of purified stem cells without differentiated or lineagecommitted cells will give short and long-term hematopoietic support(Shpall, E. J., et al. 1994, J. of Clinical Oncology 12:28-36). Sincedifferentiated cells make up a vast majority of the cells in bone marrowand blood, depletion of these cells produces a much smaller cellsuspension. The number of cells in the final product and the degree ofenrichment of progenitor/stem cells will depend on the efficiency of theantibody targeting and the removal of labeled cells.

There are several studies that enrich for hematopoietic stem cells bydepleting lineage committed cells but all require a number of positiveor negative selection steps to achieve the desired degree of enrichment(50 fold). Early studies required prior density separation and extensiveincubations to remove adherent cells (Linch, D. C, and Nathan, D. G.1984, Nature 312 20/27: 775-777; Sieff, C. A., et al., 1985, Science230: 1171-1173; Kannourakis, G. and Bol, S., 1987 Exp. Hematol15:1103-1108.). More recent techniques are no less cumbersome; involvingdensity separation steps followed by two partial lineage depletions(Winslow, J. M., et al., 1994, Bone Marrow Transplantation 14:265-271)or a partial lineage depletion using panning or FACS followed finally bypositive selection using FACS (Carlo-Stella et al. 1994, Blood 84, 10supple.:104a; Reading, C., et al. (1994), Blood 84, 10 supple.:399a).Most of these methods for lineage depletion lack effective antibodycombinations against myeloid cells, erythrocytes and/or B-cells.

U.S. Pat. Ser. No. 5,087,570 describes a process for preparing ahematopoietic cell composition using a combination of positive andnegative selection. The process relies on the use of antibody to theSca-1 antigen which is associated with murine clonogenic bone marrowprecursors of thymocytes and progeny T-cells. The Sca-1 antibody is notuseful in isolating human hematopoietic cells.

Epithelial cancers of the bronchi, mammary ducts and thegastrointestinal and urogenital tracts represent a major type of solidtumors seen today. Micrometastatic tumor cell migration is thought to bean important prognostic factor for patients with epithelial cancer(Vaughan et al., 1990, Proc. Am. Soc. Clin. Oncol. 9:9). Our ability todetect such metastatic cells is limited by the effectiveness of tissueor fluid sampling and the sensitivity of tumor detection methods. From aresearch point of view, it is also very difficult to study such rarecells and determine the biological changes which enable spread ofdisease. Metastatic epithelial tumor cells disseminate to distant sitessuch as bone marrow and lymph nodes. Bone marrow has become an importantindicator organ for the spread of epithelial cells because of its easyaccessibility and the lack of normal epithelial cells makingidentification of tumor cells less difficult. The recent trend inautologous transplantation away from the use of bone marrow grafts tocytokine mobilized peripheral blood has raised the question of how oftenperipheral blood is contaminated with micrometastatic tumor cells.Epithelial tumor contamination in peripheral blood is less frequent thanin bone marrow (Ross et al., 1993, Blood, 82(9):2605-2610) but cytokinemobilization may also “mobilize” tumor cells (Brugger et al., 1994,Blood, 83(3):636-640). Both cancer research and patient therapy couldbenefit from method of enriching epithelial tumor cells from blood, bonemarrow and peritoneal and pleural effusions.

The two most poplar methods in research laboratories for the detectionof rare epithelial tumor cells are immuno-cytochemical staining (ICC)and polyermase chain reaction (PCR). PCR detects specific DNA or RNAsequences. ICC methods rely on antibodies to epithelial-specificcytoskeleton and membrane antigens to stain tumor cells. ICC is morewidely used clinically and established laboratories with experiencedstaff are consistently reporting sensitivities of one tumor cell is 105bone marrow cells (Pantel, 1996, J. of Hematotherapy, 5:359-367). Anenrichment of 100 fold or 2 log could increase this sensitivity to onein 107 cells.

There are two approaches to enriching epithelial tumor cells from asuspension of non-epithelial cells such as bone marrow or blood. One caneither target the tumor cells for recovery using an epithelial or tumorspecific antibodies (positive selection) or target all thenon-epithelial (in this case hematopoietic cells) for depletion(negative selection). The problems with the first approach, positiveselection, is that the recovered tumor cells are covered with antibodiesand the sites commonly used for immunocytochemical detection areblocked. It is also difficult to positively select cells from samplesthat have been stored or previously frozen. The non-specific binding ofantibodies to cells or of cells to the separation matrix are too high.Negative selection, on the other hand, can deal with clumpy orpreviously frozen cell suspensions (Thomas et al., 1998, Methods inEnzymology: Signalling Pathways and Gene Regulation in HematopoieticCell Growth and Differentiation “Purification of Hematopoietic Stemcells for Further Biological Study”, Academic Press) as the recoveredcells have not been labelled with antibody. Both currently availableepithelial tumor cells enrichment methods are positive selections usingcytokeratin specific antibodies or antibodies to Human EpithelialAntigen (HEA) (Miltenyi Biotec Inc. Aubum Calif.; and Dynal, SkoyenNorway). A negative selection technique that employs antibodies to CD45has also been reported but enrichments are only 1-2 log and vary withcell source. Van Vlasselaer (U.S. Pat. No. 5,648,223) teaches aprocedure for enriching tumor cells in whole blood using cell-trapcentrifugation to enrich tumor cells in circulating bodily fluids, byseparation based on density. However, the methods taught by VanVlasselaer require the construction and operation of a cell trapcentrifuge tube calibrated to specific gradients of density, osmolalityand pH.

In order to successfully utilize circulating bodily fluids for cancerdiagnosis, improved methods of enriching the small number of circulatingtumor cells are required.

SUMMARY OF THE INVENTION

The present inventors have developed antibody compositions for use inpreparing cell preparations enriched for certain cell types such ashuman hematopoietic stem cells and progenitor cells as well asnon-hematopoietic tumor cells found in blood, bone marrow, pleural andperitoneal effusions.

To enrich for hematopoietic stem cells and progenitor cells, theantibodies in the antibody composition are specific for selected markersassociated with lineage committed or differentiated cells therebyallowing them to be removed from the cell preparation. In particular,the present inventors have found using a negative selection techniquethat an antibody composition containing antibodies specific forglycophorin A, CD3, CD24, CD16, and CD14 gives a cell preparation highlyenriched for human hematopoietic and progenitor cells. Preferably, thecomposition additionally includes antibodies to CD56, CD2, CD19, CD66eand/or CD66b. To enrich for early progenitor and stem cells(CD34^(+, CD)38⁻cells), the antibody composition also includesantibodies to CD45RA, CD36 and CD38.

The present inventors have shown that the use of the antibodycomposition of the present invention in a negative selection technique,to prepare a cell preparation which is enriched for hematopoietic stemcells and progenitor cells offers many advantages over conventionaltechniques. The antibody composition applied in one step to a sample ofperipheral blood, bone marrow, cord blood or frozen bone marrow, resultsin a greater than 50% recovery of human hematopoietic progenitor/stemcells with approximately a 3 log depletion of differentiated cells.

In addition to enriching for hematopoietic progenitor and stem cells,the above-described antibody compositions can be used to deplete tumorcells derived from hematopoietic cells such as B-cell lymphomas orT-cell leukemias. Accordingly, the present invention also provides anantibody composition to enrich for hematopoietic stem cells andprogenitor cells and to remove hematopoietic tumor cells. Thecomposition comprises antibodies specific for glycophorin A, CD3, CD24,CD16, and CD14. The composition preferably also includes antibodies toCD2, CD56, CD19, CD66e and/or CD66b.

The present invention also includes an antibody composition to enrichfor hematopoietic stem cells and progenitor cells and to removenon-hematopoietic tumor cells. In such an embodiment, the compositionalso includes antibodies specific for non-hematopoietic antigensexpressed on tumor cells, such as antibodies against antigens expressedon the surface of breast and lung carcinoma and neuroblastoma cells.Accordingly, the present invention provides an antibody composition toenrich for hematopoietic stem cells and progenitor cells and to removetumor cells comprising antibodies specific for glycophorin A, CD3, CD24,CD 16, CD14 and an antigen present on the tumor cells. The antigens onthe tumor cells is preferably a non-hematopoietic antigen expressed onthe tumor cells.

The present inventors have shown that the purging antibody compositionapplied in one step to a sample of peripheral blood, bone marrow, orfrozen bone marrow containing tumor cells, results in a greater than 50%recovery of human hematopoietic progenitor/stem cells with approximatelya 3-5 log depletion of tumor cells.

The high level of enrichment obtained using the antibody compositions ofthe invention, does not require additional enrichment or tumor purgingsteps, which would result in loss of, or damage to, progenitor and stemcells. The recovery of CD34⁺ cells, CD34⁺CD38⁻ cells, colony formingcells, and LTCIC, is also much higher than with conventional multisteptechniques.

The present inventors have also developed an antibody composition foruse in preparing cell preparations enriched for non-hematopoietic tumorcells, in particular metastatic tumor cells. The composition is usefulin the detection of non-hematopoietic tumor cells from blood and bonemarrow of patients to aid in the detection of metastatic disease. Thetumor-enriching antibody composition contains antibodies specific forselected markers associated with hematopoietic cells. In particular, thepresent inventors have found using a negative selection technique thatan antibody composition containing antibodies specific for glycophorinA, CD2, CD14, CD16, CD38, CD45 and CD66b, and optionally CD3, CD36,CD56, and/or CD66e, gives a cell preparation highly enriched fornon-hematopoietic tumor cells. The present inventors have shown that thetumor enriching antibody compositions applied in one step to a sample ofperipheral blood, frozen peripheral blood, bone marrow or pleural orperitoneal effusions containing tumor cells results at least a 2 logenrichment (and typically greater than 3 log enrichment), of the tumorcells.

The enrichment and recovery of human hematopoietic progenitor and stemcells as well as non-hematopoietic tumor cells using the antibodycompositions of the invention in a negative selection technique has manyadvantages over conventional positive selection techniques. As mentionedabove, highly enriched cell preparations can be obtained using a singlestep. The cells obtained using the antibody composition of the inventionare not labeled or coated with antibodies or modified making them highlysuitable for many uses. For example, the isolated hematopoietic stemcells and progenitor cells can be used in transplantation and othertherapeutic uses. The isolated metastatic tumor cells can be used todetect metastatic disease in blood and bone marrow as well as pleuraland peritoneal effusions.

The present invention also relates to a negative selection process forenriching and recovering human hematopoietic progenitor cells and stemcells in a sample containing human hematopoietic differentiated,progenitor, and stem cells comprising (a) reacting the sample with anantibody composition containing antibodies capable of binding to theantigens glycophorin A, CD3 CD24, CD16, and CD14, and optionally CD2,CD56, CD19, CD66e and/or CD66b under conditions permitting the formationof conjugates between the antibodies and cells in the sample having theantigens glycophorin A, CD3 CD24, CD16, and CD14, and optionally CD2,CD56, CD19, CD66e and/or CD66b on their surfaces; (b) removing theconjugates; and (c) recovering a cell preparation which is enriched inhuman hematopoietic progenitor cells and stem cells.

The present invention further provides a negative selection process forenriching and recovering normal human hematopoietic progenitor cells andstem cells and depleting hematopoietic tumor cells in a samplecontaining human hematopoietic differentiated, progenitor, and stemcells, and hematopoietic tumor cells comprising (a) reacting the samplewith an antibody composition containing antibodies capable of binding tothe antigens glycophorin A, CD3, CD24, CD16, CD14, and optionally CD2,CD56, CD19, CD66e and/or CD66b, under conditions so that conjugates areformed between the antibodies and the cells in the sample having theantigens glycophorin A, CD3 CD24, CD16, and CD14, and optionally CD2,CD56, CD19, CD66e and/or CD66b; (b) removing the conjugates; and (c)recovering a cell preparation which is enriched in normal humanhematopoietic progenitor cells and stem cells and depleted inhematopoietic tumor cells.

The present invention further provides a process for enriching andrecovering human hematopoietic stem cells and progenitor cells anddepleting tumor cells in a sample containing differentiated cells,progenitor cells, stem cells and tumor cells, comprising (a) reactingthe sample with an antibody composition containing antibodies capable ofbinding to the antigens glycophorin A, CD3 CD24, CD16, and CD14, and anantigen present on the tumor cells and optionally CD2, CD56, CD19, CD66eand/or CD66b under conditions permitting the formation of conjugatesbetween the antibodies and cells in the sample having the antigensglycophorin A, CD3 CD24, CD16, and CD14, and an antigen present on thetumor cells and optionally CD2, CD56, CD19, CD66e and/or CD66b on theirsurfaces; (b) removing the conjugates; and (c) recovering a cellpreparation which is enriched in human hematopoietic progenitor cellsand stem cells and depleted in tumor cells.

The present invention also contemplates a negative selection process forenriching for non-hematopoietic metastatic tumor cells in a samplecontaining the tumor cells and hematopoietic cells comprising (a)reacting the sample with an antibody composition comprising antibodiesspecific for glycophorin A, CD2, CD14, CD16, CD38, CD45 and CD66b, andoptionally CD3, CD36, CD56 and/or CD66e under conditions so thatconjugates are formed between the antibodies and hematopoietic cells inthe sample expressing the antigens glycophorin A, CD2, CD14, CD16, CD38,CD45 and CD66b, and optionally CD3, CD36, CD56 and/or CD66e; (b)removing the conjugates; and (c) recovering a cell preparation enrichedin the tumor cells.

The present invention also relates to a kit useful in preparing a cellpreparation enriched in human hematopoietic progenitor and stem cellscomprising antibodies specific for glycophorin A, CD3, CD24, CD16, andCD14, and instructions for preparing a cell preparation enriched inhematopoietic progenitor and stem cells.

The present invention further includes a kit useful in preparing a cellpreparation enriched in hematopoietic stem cells and progenitor cellsand depleted in hematopoietic tumor cells comprising antibodies specificfor glycophorin A, CD3, CD24, CD16, and CD14 and instructions forpreparing a cell preparation enriched in hematopoietic stem cells andprogenitor cells and depleted in hematopoietic tumor cells.

The present invention also includes a kit useful in preparing a cellpreparation enriched in hematopoietic stem cells and progenitor cellsand depleted in tumor cells comprising antibodies specific forglycophorin A, CD3, CD24, CD16, CD14, and an antigen present on thetumor cells and instructions for preparing a cell preparation enrichedin hematopoietic stem cells and progenitor cells and depleted in tumorcells.

The present invention also relates to a kit useful in preparing a cellpreparation enriched in non-hematopoietic tumor cells from blood, bonemarrow, pleural or peritoneal effusions, comprising antibodies specificfor glycophorin A, CD2, CD14, CD16, CD38, CD45 and CD66b, andinstructions for preparing a cell preparation enriched innon-hematopoietic tumor cells.

The invention further relates to cell preparations obtained inaccordance with the processes of the invention. The invention stillfurther contemplates a method of using the antibody compositions of theinvention in negative selection methods to recover a cell preparationwhich is enriched in human hematopoietic progenitor and stem cells ornon-hematopoietic tumor cells.

These and other aspects of the present invention will become evidentupon reference to the following detailed description and attacheddrawings. In addition, reference is made herein to various publications,which are hereby incorporated by reference in their entirety.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will now be described with reference to the accompanyingdrawings, in which:

FIG. 1 is a schematic representation of magnetic cell labeling usingtetrameric antibody complexes and colloidal dextran iron; and

FIG. 2A shows a Fluorescence Activated Cell Sorting (FACS) histogram ofmobilized peripheral blood before progenitor enrichment using theprogenitor enrichment composition.

FIG. 2B shows a Fluorescence Activated Cell Sorting (FACS) histogram ofmobilized peripheral blood after progenitor enrichment using theprogenitor enrichment composition.

FIG. 3A shows a Fluorescence Activated Cell Sorting (FACS) profile ofmobilized peripheral blood before enrichment using the primitiveprogenitor enrichment composition.

FIG. 3B shows a Fluorescence Activated Cell Sorting (FACS) profile ofmobilized peripheral blood after enrichment using the primitiveprogenitor enrichment composition.

FIG. 4A shows a Fluorescence Activated Cell Sorting (FACS) profile ofperipheral blood seeded with CAMA Breast carcinoma cell line beforeenrichment using the tumor enrichment composition.

FIG. 4B shows a Fluorescence Activated Cell Sorting (FACS) profile ofperipheral blood seeded with CAMA Breast carcinoma cell line afterenrichment using the tumor enrichment composition.

FIG. 5A shows a Fluorescence Activated Cell Sorting (FACS) profile ofperipheral blood seeded with pleural effusion cells before enrichmentusing the tumor enrichment composition.

FIG. 5B shows a Fluorescence Activated Cell Sorting (FACS) profile ofperipheral blood seeded with pleural effusion cells after enrichmentusing the tumor enrichment composition.

DETAILED DESCRIPTION OF THE INVENTION I. HEMATOPOIFTIC CELL TYPES ANDTUMOR CELLS

The term “differentiated cells” used herein refers to humanhematopoietic cells which have limited or no proliferative capacity.Differentiated cells represent specialized end cells that are found inblood, and their immediate precursors.

The term “progenitor cells” used herein refers to cells which are theimmediate precursors of the differentiating cells. Most of theprogenitor cells differentiate along a single lineage but they may havequite extensive proliferative capacity. Progenitor cells appearmorphologically as blast cells, and they typically do not have specificfeatures of the hematopoietic lineage to which they are committed.

The term “stem cells” used herein refers to the cells from whichprogenitor cells are derived. Stem cells are defined by their ability toself-renew as well as to generate daughter cells of any of thehematopoietic lineages. Stem cells with long term hematopoieticreconstituting ability can be distinguished by a number of physical andbiological properties from differentiated cells and progenitor cells(Hodgson, G. S. & Bradley, T. R., Nature, Vol. 281, pp. 381-382; Visseret al., 1984, J. Exp. Med., Vol. 59, pp. 1576-1590; Spangrude et al.,1988, Science, Vol. 241, pp. 58-62; Szilvassy et al., 1989, Blood, Vol.74, pp. 930-939; Ploemacher, R. E. & Brons, R. H. C., 1989, Exp.Hematol., Vol. 17, pp. 263-266).

The presence of stem cells and progenitor cells in a cell preparationmay be detected by their ability to produce colony-forming cells inculture or to repopulate xeonogenic hosts such as immunodeficient mice.They may also be detected by screening for the CD34 antigen which is apositive marker for early hematopoietic cells including colony formingcells and stem cells. Primitive hematopoietic stem cells with long termhematopoietic reconstituting ability can be identified by determiningthe number of clonogenic cells present after 5 to 8 weeks in long termcultures (Sutherland et al., 1986, Blood, Vol. 74, p. 1563; Udomsakdi etal., 1991, Exp. Hematol., Vol. 19, p. 338; and, Sutherland et al., 1990,Proc. Natl. Acad. Sci., Vol. 87, p. 3584).

Tumor cells which may be removed from a sample using the antibodycompositions and processes described herein include tumor cells whichhave non-hematopoietic antigens or markers expressed on their surfacesi.e. antigens that distinguish the tumor cells from hematopoieticprogenitor cells and stem cells. For example, specific markers have beenfound to be expressed on tumor cells such as breast and lung carcinoma,and neuroblastoma. Table 4 lists specific examples of antibodies whichrecognize non-hematopoietic antigens expressed on tumor cells.

Some metastatic tumor cells express hematopoietic lineage markers orantigens, for example, tumor cells from B-lymphomas, multiple myeloma,some chronic lymphocytic leukemias (CLL), and some acute lymphocyticleukemias (ALL) express B-cell markers such as CD22, CD20, CD29, and Tcells from ALL and CLL express T-cell markers, and antibodies to theseantigens may be included in the antibody compositions of the inventionto remove tumor cells expressing the hematopoietic lineage antigens.

Tumor cells which may be enriched in a sample using the antibodycompositions and processes described herein include non-hematopoietictumor cells which do not express hematopoietic lineage markers.Non-hematopoietic tumors include epithelial cancers of the bronchi,mammary ducts, gastrointestinal tract, reproductive system andurogenital tract such as carcinomas of the lung, breast, colon,prostate, bladder, ovary, endometrium, cervix, pancreas, oesophagus,small bowel, rectum, uterus, stomach, larynx, skin and vagina.

II. ANTIBODY COMPOSMONS

As hereinbefore mentioned, the invention relates to an antibodycompositions for preparing enriched cell preparations. In one aspect,the antibody composition is for enriching human hematopoieticprogenitors and stem cells and comprises antibodies specific for theantigens glycophorin A, CD3, CD24, CD16, and CD14, which are present onthe surface of human differentiated cells. In a preferred embodiment,the antibody composition further includes antibodies to CD2, CD56, CD19,CD66e and/or CD66b. The composition may also include antibodies toCD45RA, CD38, and/or CD36.

The antibody composition for enriching for human hematopoieticprogenitor and stem cells may be generally referred to as the“progenitor enrichment composition” or the “progenitor enrichmentcocktail”. One skilled in the art will appreciate that in addition tothe antibodies listed above, the progenitor enrichment cocktail mayadditionally include other antibodies that are specific for antigens onthe surface of differentiated cells including those listed in Table 2.The selection of the antibodies can depend on many factors including thenature of the sample to be enriched. In an embodiment of the invention,an antibody composition is provided for enriching and recovering humanhematopoietic progenitor and stem cells from fresh bone marrowconsisting of antibodies specific for glycophorin A, CD3, CD24, CD16,CD14, CD66e and CD66b. In a second embodiment, an antibody compositionis provided for enriching and recovering human hematopoietic progenitorand stem cells from previously frozen bone marrow consisting ofantibodies specific for glycophorin A, CD3, CD24, CD16, and CD14. In afurther embodiment of the invention, an antibody composition is providedfor enriching and recovering human hematopoietic progenitor and stemcells from peripheral or cord blood consisting of antibodies specificfor glycophorin A, CD3, CD24, CD16, CD14, CD66e, CD66b, CD56, CD2 andCD19.

Pluripotent stem cells and committed progenitors express CD34, and thisCD34 compartment can be subdivided using antibodies to a variety of cellsurface markers. Stem cells co-purify in a population of CD34⁺ cellswhich lack or have low expression of certain lineage markers (CD38,CD33, CD45RA, CD71, CD36 and HLA-DR) (Craig et al. 1994, British Journalof Haematology, 88:24-30; Lansdorp, P. AI. and Dragowska, W. 1992 J.Exp. Med. 175:1501-1509; Sutherland, H, J., et al. 1989 Blood74.1563-1570). Antibodies recognizing these antigens can be included inthe antibody composition to further enrich for stem cells, while losingsome of the committed mature CD34⁺ cells. Preferably, anti-CD45RA,anti-CD38 and anti-CD36 are included in the antibody composition.Accordingly, in another embodiment the present invention provides anantibody composition for enriching for early progenitor cells comprisingantibodies specific for glycophorin A, CD3, CD24, CD16, CD14, CD2, CD56,CD19, CD66b, CD45RA, CD36 and CD38.

In another aspect, the present invention also relates to an antibodycomposition for enriching and recovering human hematopoietic progenitorand stem cells and depleting hematopoietic tumor cells comprisingantibodies specific for glycophorin A, CD3, CD24, CD16 and CD14. In apreferred embodiment the antibody composition further includesantibodies to CD2, CD56, CD19, CD66e and/or CD66b.

The present invention also includes an antibody composition forenriching and recovering hematopoietic stem cells and progenitor cellsand depleting non-hematopoietic tumor cells. In such an embodiment, thecomposition also includes antibodies specific for non-hematopoieticantigens expressed on tumor cells, such as antibodies against antigensexpressed on the surface of breast and lung carcinoma and neuroblastomacells. The antibodies to the tumor antigens may be obtained fromcommercial sources or prepared using techniques known in the art.Preferably, the antibodies specific for non-hematopoietic antigens arespecific for antigens expressed on breast and lung carcinoma andneuroblastoma cells, for example a shown in Table 4.

In a further aspect, the invention also includes an antibody compositionfor enriching and recovering non-hematopoietic tumor cells from blood,bone marrow, pleural and peritoneal effusions comprising antibodiesspecific for glycophorin A, CD2, CD14, CD16, CD38, CD45 and CD66b, andoptionally CD3, CD36, CD56 and/or CD66e.

Within the context of the present invention, antibodies are understoodto include monoclonal antibodies and polyclonal antibodies, antibodyfragments (e.g., Fab, and F(ab′)₂) and chimeric antibodies. Antibodiesare understood to be reactive against a selected antigen on the surfaceof a differentiated cell or tumor cell if they bind with an appropriateaffinity (association constant), e.g. greater than or equal to 10⁷ M⁻¹.

Polyclonal antibodies against selected antigens on the surface ofdifferentiated cells or tumor cells may be readily generated by one ofordinary skill in the art from a variety of warm-blooded animals such ashorses, cows, various fowl, rabbits, mice, hamsters, or rats. Forexample, a mammal, (e.g., a mouse, hamster, or rabbit) can be immunizedwith an immunogenic form of an antigen which elicits an antibodyresponse in the mammal. Techniques for conferring immunogenicity on anantigen include conjugation to carriers or other techniques well knownin the art. For example, the antigen can be administered in the presenceof adjuvant. The progress of immunization can be monitored by detectionof antibody titers in plasma or serum. Following immunization, antiseracan be obtained and polyclonal antibodies isolated from the sera.

Monoclonal antibodies are preferably used in the antibody compositionsof the invention. Monoclonal antibodies specific for selected antigenson the surface of differentiated cells or tumor cells may be readilygenerated using conventional techniques. For example, monoclonalantibodies may be produced by the hybridoma technique originallydeveloped by Kohler and Milstein 1975 (Nature 256, 495-497; see alsoU.S. Pat. Nos. RE 32,011, 4,902,614, 4,543,439, and 4,411,993 which areincorporated herein by reference; see also Monoclonal Antibodies,Hybridomas: A New Dimension in Biological Analyses, Plenum Press,Kennett, McKeam, and Bechtol (eds.), 1980, and Antibodies: A LaboratoryManual, Harlow and Lane (eds.), Cold Spring Harbor Laboratory Press,1988). Other techniques may also be utilized to construct monoclonalantibodies (for example, see William D. Huse et al., 1989, “Generationof a Large Combinational Library of the Immunoglobulin Repertoire inPhage Lambda,” Science 246:1275-1281, L. Sastry et al., 1989 “Cloning ofthe Immunological Repertoire in Escherichia coli for Generation ofMonoclonal Catalytic Antibodies: Construction of a Heavy Chain VariableRegion-Specific cDNA Library,” Proc Natl. Acad. Sci USA 86:5728-5732;Kozbor et al., 1983 Immunol. Today 4, 72 re the human B-cell hybridomatechnique; Cole et al. 1985 Monoclonal Antibodies in Cancer Therapy,Allen R. Bliss, Inc., pages 77-96 re the EBV-hybridoma technique toproduce human monoclonal antibodies; and see also Michelle Alting-Meeset al., 1990 “Monoclonal Antibody Expression Libraries: A RapidAlternative to Hybridomas,” Strategies in Molecular Biology 3:1-9).Hybridoma cells can be screened immunochemically for production ofantibodies specifically reactive with an antigen, and monoclonalantibodies can be isolated.

The term “antibody” as used herein is intended to include antibodyfragments which are specifically reactive with specific antigens on thesurface of differentiated cells or tumor cells. Antibodies can befragmented using conventional techniques and the fragments screened forutility in the same manner as described above for whole antibodies. Forexample, F(ab′)₂ fragments can be generated by treating antibody withpepsin. The resulting F(ab′)₂ fragment can be treated to reducedisulfide bridges to produce Fab′ fragments.

The invention also contemplates chimeric antibody derivatives, i.e.,antibody molecules that combine a non-human animal variable region and ahuman constant region. Chimeric antibody molecules can include, forexample, the antigen binding domain from an antibody of a mouse, rat, orother species, with human constant regions. A variety of approaches formaking chimeric antibodies have been described and can be used to makechimeric antibodies containing the immunoglobulin variable region whichrecognizes selected antigens on the surface of differentiated cells ortumor cells. See, for example, Morrison et al., 1985; Proc. Natl. Acad.Sci. U.S.A. 81,6851; Takeda et al., 1985, Nature 314:452; Cabilly etal., U.S. Pat. No. 4,816,567; Boss et al., U.S. Pat. No. 4,816,397;Tanaguchi et al., European Patent Publication EP171496; European PatentPublication 0173494, United Kingdom patent GB 2177096B.

Antibodies against selected antigens on the surface of differentiatedcells or tumor cells may also be obtained from commercial sources asillustrated in Tables 2 and 4.

Antibodies may be selected for use in the antibody compositions of theinvention based on their ability to deplete targeted differentiatedcells and/or tumor cells and recover non-targeted cells (i.e. normalprogenitor and stem cells, or specific differentiated cells) in magneticcell separations as more particularly described herein, and in U.S. Pat.No. 5,514,340, which is incorporated in its entirety herein byreference. In general, an antibody is selected that gives greater than 3log depletion of differentiated cells or tumor cells, with greater than75% recovery of CD34⁺ cells (bone marrow, mobilized blood and cordblood) or non-targeted lymphocytes (steady state blood), in testmagnetic cell separations as described herein.

The anti-glycophorin A antibodies contained in the antibody compositionof the invention are used to label erythrocytes. Examples of monoclonalantibodies specific for glycophorin A are 2B7.1 (StemCell Technologies),10F7MN (U.S. Pat. No. 4,752,582, Cell lines: ATCC accession numbersHB-8162), and D2.10 (Immunotech, Marseille, France). The concentrationof antiglycophorin A antibodies used in the antibody composition aregenerally less than the concentration that will cause agglutination(i.e. 3-10 μg/ml). Preferably the concentration of antiglycophorin Aantibodies used in the antibody composition is between about 0.5 to 5μg/ml, preferably 1 to 2 μg/ml.

Monoclonal antibodies against CD24, CD3, CD19, CD20, CD22, CD29, CD56,CD2 in the antibody composition of the invention are used to label B andT lymphocytes and NK cells. Examples of monoclonal antibodies specificfor CD24, CD3, CD19, CD20, CD22, CD56, and CD2, are 32D12 (Dr. SteinarFunderud, Institute for Cancer Research, Dept. of Immunology, Oslo,Norway,) and ALB9 (Immunotech, Marseille, France); UCHT1 (Immunotech,Marseille, France) and SK7 (Becton Dickinson, Mountain View, Calif.);J4.119 (Immunotech, Marseille, France) and Leu-12 (Becton Dickinson,Mountain View, Calif.); MEM97 (Dr. Horejsi, Institute of MolecularGenetics Academy of Sciences of the Czech Republic, Praha, CzechRepublic, or Cedarlane Laboratories, Homby, Ontario, Canada) and Leu-16(Becton Dickinson, Mountain View, Calif.); SJ10.1H11 (Immunotech,Marseille, France); T199 (Immunotech, Marseille, France); and 6F10.3(Immunotech, Marseille, France), respectively. The concentration of eachof the monoclonal antibodies against CD24, CD3, CD19, CD20, CD56, CD2contained in the antibody composition is between about 0.5 to 5 μg/ml,preferably 2 to 3 μg/ml.

Monoclonal antibodies against CD14, CD16, CD66e and CD66b in theantibody compositions of the invention are used to label monocytes andgranulocytes. Examples of monoclonal antibodies specific for CD14, CD16,CD66e and CD66b, are MEM15 and MEM18 (Dr. Vaclav Horejsi, Institute ofMolecular Genetics Academy of Sciences of the Czech Republic, Praha,Czech Republic; Cedarlane Laboratories, Homby, Ontario, Canada); MEM154(Dr. Vaclav Horejsi, Institute of Molecular Genetics Academy of Sciencesof the Czech Republic, Praha, Czech Republic; Cedarlane Laboratories,Hornby, Ontario, Canada), Leu-lla (Becton Dickinson, Mountain View,Calif.), and 3G8 (Immunotech, Marseille, France); CLB/gran10 (CLB,Central Laboratory of the Netherlands, Red Cross Blood TransfusionService); and, B13.9 (CLB, Central Laboratory of the Netherlands, RedCross Blood Transfusion Service) and 80H3 (Immunotech, Marseille,France), respectively. The concentration of each of the monoclonalantibodies against CD14, CD16, CD66e and CD66b contained in the antibodycomposition is between about 0.5 to 5 μg/ml, preferably 2-3 μg/ml.

Monoclonal antibodies against CD45RA, CD38 and CD36 are used to labelT-cells, B-cells plasma cells, granulocytes, platelets, monocytes,differentiated erythroid precursors, and some committed matureprogenitors, to further enrich for stem cells. Examples of monoclonalantibodies against CD45RA, CD38 and CD36 are 8D2.2 (StemCellTechnologies, Vancouver, Canada, Craig et al., 1994, British Journal ofHaematology, 88:24-30.), Leu-18 (Becton Dickinson, Mountain View,Calif.); T16 (Immunotech, Marseille, France); and, FA60152 (Immunotech,Marseille, France) and IVC7 (CLB, Central Laboratory of the NetherlandsRed Cross Blood Transfusion Service), respectively. The concentration ofeach of the monoclonal antibodies against CD45RA and CD36 contained inthe antibody composition is between about 0.5 to 5 μg/ml, preferably 1to 3 μg/ml.

Table 2 sets out the most preferred monoclonal antibodies specific fordifferentiated cells, their sources and concentrations, for use in theantibody compositions of the invention. Table 4 sets out the mostpreferred monoclonal antibodies specific for tumor cells, and commercialsources/references for the antibodies.

In one embodiment of the invention the antibody composition forenriching for hematopoietic stem cells and progenitor cells, comprises2B7.1 (glycophorin A), SK7 (CD3), 32D12 (CD24), MEM54 (CD16), and MEM15(CD14).

In another embodiment of the invention the antibody composition forenriching for hematopoietic stem cells and progenitor cells, comprises2B7.1 (glycophorin A), SK7 (CD3), 32D12 (CD24), MEM54 (CD16), MEM15(CD14), 6F10.2 (CD2), T199 (CD56), J4.119 (CD19) and/or 80H3 (CD66b).

In further embodiment of the invention the antibody composition forenriching for early hematopoietic stem and progenitor cells comprisesthe monoclonal antibodies designated 2B7.1 (glycophorin A), SK7 (CD3),MEM15 (CD14), MEM154 (CD16), 32D12 (CD24), 80H3 (CD66b), J4.119 (CD19),6F10.3 (CD2), MY31 (CD56), 8D2.2 (CD45RA), T16 (CD38) and FA60152(CD36), or comprises the monoclonal antibodies designated 10F7MN(glycophorin A), UCHT1 (CD3), ALB9 (CD24), 3G8 (CD16), MEM15 (CD14),B13.9 (CD66b), T199 (CD56), 6F10.3 (CD2), J4.119 (CD19), 8D2.2 (CD45RA),T16 (CD38) and 1VC7 (CD36).

A preferred antibody composition for removing differentiatedhematopoietic cells and breast and lung carcinoma cells from a samplecomprises the monoclonal antibodies 2B7.1 (glycophorin A), SK7 (CD3),MEM15 (CD14), 3G8 (CD16), ALB9 (CD24), 80H3 (CD66b), J4.119 (CD19),6F10.3 (CD2), MY31 (CD56), or the monoclonal antibodies 1OF7MN(glycophorin A), SK7 (CD3), 32D12 (CD24), MEM154 (CD16), MEM15 (CD14),80H3 (CD66b) or B13.9 (CD66b), T199 (CD56), 6F10.3 (CD2), J4.119 (CD19),and one or more of the monoclonal antibodies specific for an antigen onthe surface of a breast or lung carcinoma as set forth in Table 4. Mostpreferably the monoclonal antibodies specific for an antigen on thesurface of cells from a breast carcinoma used in a composition of theinvention are one or more of 5E11, H23A, 6E7, RAR, BerEp4 and BRST1.

Preferred antibody compositions for enriching for non-hematopoieticmetastatic tumor cells from a sample containing hematopoietic cells andnon-hematopoietic metastatic tumor cells comprise the monoclonalantibodies 2B7.1 (glycophorin A); MEM15 (CD14); 3G8 (CD16); 80H3(CD66b); 6F10.3 (CD2); T16 (CD38); and MEM28 (CD45).

Antibody compositions in accordance with the present invention may beprepared which lack antibodies to a specific differentiated cell type orlineage committed cell. For example, an antibody composition may beprepared which does not contain antibodies to the CD14, and CD16antigens which are expressed on monocytes. This composition may be usedto prepare a cell preparation which is enriched for monocytes. Otherexamples of antibody compositions which can be used to prepare cellpopulations enriched for monocytes, B-cells, T-cells, CD4⁺ T-cells, CD8⁺T-cells, and NK cells are set out in Table 3.

III. PROCESS FOR PREPARING ENRICHED CELL PREPARATIONS

The antibody compositions of the invention may be used to enrich andrecover cell preparations enriched in a specific cell type such as stemcells and progenitor cells or non-hematopoietic tumor cells. Inaccordance with a process of the invention, a sample is reacted with anantibody composition containing antibodies which are specific forselected antigens on the surface of the cells to be removed from thesample and not on the cells to be enriched in the sample, under suitableconditions, conjugates form between the antibodies contained in theantibody composition and the cells in the sample containing the antigenson their surface; and the conjugates are removed to provide a cellpreparation enriched in specific cells.

(a) Progenitor Cell Enrichment

In one aspect the present invention provides a negative selectionprocess for enriching and recovering human hematopoietic progenitorcells and stem cells in a sample containing human hematopoieticdifferentiated, progenitor, and stem cells comprising (a) reacting thesample with an antibody composition containing antibodies capable ofbinding to the antigens glycophorin A, CD3 CD24, CD16, and CD14 underconditions so that conjugates are formed between the antibodies andcells in the sample containing the antigens glycophorin A, CD3 CD24,CD16, and CD14 on their surfaces; (b) removing the conjugates; and, (c)recovering a cell preparation which is enriched in human hematopoieticprogenitor cells and stem cells.

The antibody composition for enriching for progenitor and stem cells mayadditionally include other antibodies specific for antigens ondifferentiated cells such as CD2, CD56, CD19, CD66e and/or CD66b. Theselection of antibodies can largely depend on the nature of the sampleto be enriched. When the sample is fresh bone marrow, the compositionpreferably comprises antibodies to glycophorin A, CD3, CD24, CD16, CD14,CD66e and CD66b. When the sample is mobilized peripheral blood or cordblood, the composition preferably comprises glycophorin A, CD3, CD24,CD16, CD14, CD66e, CD66b, CD56, CD2, and CD19. To enrich for earlyprogenitor cells the composition preferably includes glycophorin A, CD3,CD24, CD16, CD14, CD66e, CD66b, CD56, CD2, CD19, CD45RA, CD36 and CD38.

The inventors have demonstrated that their method of progenitorenrichment provides a cell preparation with greater than 50% recovery ofprogenitor and stem cells and 3 long depletion of differentiated cells.

(b) Tumor Cell Depletion

In another aspect, the present invention provides a negative selectionprocess for enriching and recovering normal human hematopoieticprogenitor cells and stem cells and depleting hematopoietic tumor cellsin a sample containing human hematopoietic differentiated, progenitor,and stem cells, and tumor cells comprising (a) reacting the sample withan antibody composition containing antibodies capable of binding to theantigens glycophorin A, CD3, CD24, CD16, and CD14, under conditions sothat conjugates are formed between the antibodies and the cells in thesample having the antigens glycophorin A, CD3 CD24, CD16 and CD14; (b)removing the conjugates; and (c) recovering a cell preparation which isenriched in normal human hematopoietic progenitor cells and stem cellsand depleted in hematopoietic tumor cells.

The present invention further provides a process for enriching andrecovering human hematopoietic stem cells and progenitor cells anddepleting tumor cells in a sample containing differentiated cells,progenitor cells, stem cells and tumor cells, comprising (a) reactingthe sample with an antibody composition containing antibodies capable ofbinding to the antigens glycophorin A, CD3 CD24, CD16, and CD14, and anantigen present on the tumor cells and optionally CD2, CD56, CD19, CD66eand/or CD66b under conditions permitting the formation of conjugatesbetween the antibodies and cells in the sample having the antigensglycophorin A, CD3 CD24, CD16, and CD14, and an antigen present on thetumor cells and optionally CD2, CD56, CD19, CD66e and/or CD66b on theirsurfaces; (b) removing the conjugates; and (c) recovering a cellpreparation which is enriched in human hematopoietic progenitor cellsand stem cells and depleted in tumor cells.

The inventors have demonstrated that the method of tumor cell depletionprovides a cell preparation with a 3 log depletion of tumor cells.

(c) Tumor Cell Enrichment

In another aspect, the present invention provides a negative selectionprocess for enriching for non-hematopoietic metastatic tumor cells in asample containing the tumor cells and hematopoietic cells comprising (a)reacting the sample with an antibody composition comprising antibodiesspecific for glycophorin A, CD2, CD14, CD16, CD38, CD45 and CD66b underconditions so that conjugates are formed between the antibodies andhematopoietic cells in the sample expressing the antigens glycophorin A,CD2, CD14, CD16, CD38, CD45 and CD66b; (b) removing the conjugates; and(c) recovering a cell preparation enriched in the tumor cells.

In one embodiment, the tumor cells are metastatic tumor cells derivedfrom epithelial cancers of the bronchi, mammary ducts, reproductivesystem, gastrointestinal tract and urogenital tract such as lungcarcinoma, breast carcinoma, colon carcinoma, prostate carcinoma andbladder carcinoma.

The inventors have demonstrated that their method of tumor cellenrichment provides a cell preparation that is enriched at least 2 log,generally 3-4 log, in tumor cells. The tumor enriched cell preparationscan be used to detect metastatic tumor cells in sample. Accordingly, thepresent invention also provides a method of detecting tumor metastasisin a sample comprising (a) reacting the sample with an antibodycomposition comprising antibodies specific for glycophorin A, CD2, CD14,CD16, CD38, CD45 and CD66b under conditions so that conjugates areformed between the antibodies and hematopoietic cells in the sampleexpressing the antigens glycophorin A, CD2, CD14, CD16, CD38, CD45 andCD66b; (b) removing the conjugates; and (c) recovering a cellpreparation enriched in the tumor cells; and (d) detecting the tumorcells in the cell preparation. The tumor cells may be detected usingtechniques known in the art. For example, antibodies specific for tumorcells may be used in antibody mediated detection methods such asimmuno-cytochemical staining (ICC).

In all of the above negative selection processes for cell enrichment,conditions which permit the formation of conjugates may be selectedhaving regard to factors such as the nature and amounts of theantibodies in the antibody composition, and the estimated concentrationof targeted cells in the sample.

The antibodies in the antibody compositions may be labelled with amarker or they may be conjugated to a matrix. Examples of markers arebiotin, which can be removed by avidin bound to a support, andfluorochromes, e.g. fluorescein, which provide for separation usingfluorescence activated sorters. Examples of matrices are magnetic beads,which allow for direct magnetic separation (Kernshead 1992), panningsurfaces e.g. plates, (Lebkowski, J. S, et al., (1994), J. of CellularBiochemistry supple. 18b:58), dense particles for density centrifugation(Van Vlasselaer, P., Density Adjusted Cell Sorting (DACS), A NovelMethod to Remove Tumor Cells From Peripheral Blood and Bone MarrowStemCell Transplants. (1995) 3rd International Symposium on RecentAdvances in Hematopoietic Stem Cell Transplantation-Clinical Progress,New Technologies and Gene Therapy, San Diego, Calif.), dense particlesalone (Zwemer et al., Immunol. Meth. 1996 198(2):199-202) adsorptioncolumns (Berenson et al. 1986, Journal of Immunological Methods91:11-19.), and adsorption membranes. The antibodies may also be joinedto a cytotoxic agent such as complement or a cytotoxin, to lyse or killthe targeted differentiated or tumors cells.

The antibodies in the antibody compositions may be directly orindirectly coupled to a matrix. For example, the antibodies in thecompositions of the invention may be chemically bound to the surface ofmagnetic particles for example, using cyanogen bromide. When themagnetic particles are reacted with a sample, conjugates will formbetween the magnetic particles with bound antibodies specific forantigens on the surfaces of the differentiated cells and/or tumor cells,and the differentiated cells and/or tumor cells having the antigens ontheir surfaces.

Alternatively, the antibodies may be indirectly conjugated to a matrixusing antibodies. For example, a matrix may be coated with a secondantibody having specificity for the antibodies in the antibodycomposition. By way of example, if the antibodies in the antibodycomposition are mouse IgG antibodies, the second antibody may be rabbitanti-mouse IgG.

The antibodies in the antibody compositions may also be incorporated inantibody reagents which indirectly conjugate to a matrix. Examples ofantibody reagents are bispecific antibodies, tetrameric antibodycomplexes, and biotinylated antibodies.

Bispecific antibodies contain a variable region of an antibody in anantibody composition of the invention, and a variable region specificfor at least one antigen on the surface of a matrix. The bispecificantibodies may be prepared by forming hybrid hybridomas. The hybridhybridomas may be prepared using the procedures known in the art such asthose disclosed in Staerz & Bevan, (1986, PNAS (USA) 83: 1453) andStaerz & Bevan, (1986, Immunology Today, 7:241). Bispecific antibodiesmay also be constructed by chemical means using procedures such as thosedescribed by Staerz et al., (1985, Nature, 314:628) and Perez et al.,(1985 Nature 316:354), or by expression of recombinant immunoglobulingene constructs.

A tetrameric immunological complex may be prepared by mixing a firstmonoclonal antibody which is capable of binding to at least one antigenon the surface of a matrix, and a second monoclonal antibody from theantibody composition of the invention. The first and second monoclonalantibody are from a first animal species. The first and second antibodyare reacted with an about equimolar amount of monoclonal antibodies of asecond animal species which are directed against the Fc-fragments of theantibodies of the first animal species. The first and second antibodymay also be reacted with an about equimolar amount of the F(ab′)₂fragments of monoclonal antibodies of a second animal species which aredirected against the Fc-fragments of the antibodies of the first animalspecies. (See U.S. Pat. No. 4,868,109 to Lansdorp, which is incorporatedherein by reference for a description of tetrameric antibody complexesand methods for preparing same).

The antibodies of the invention may be biotinylated and indirectlyconjugated to a matrix which is labelled with (strept) avidin. Forexample, biotinylated antibodies contained in the antibody compositionof the invention may be used in combination with magnetic iron-dextranparticles that are covalently labelled with (strept) avidin (Miltenyi,S. et al., Cytometry 11:231, 1990). Many alternative indirect ways tospecifically cross-link the antibodies in the antibody composition andmatrices would also be apparent to those skilled in the art.

In an embodiment of the invention, the cell conjugates are removed bymagnetic separation using magnetic particles. Suitable magneticparticles include particles in ferr6fluids and other colloidal magneticsolutions. “Ferrofluid” refers to a colloidal solution containingparticles consisting of a magnetic core, such as magnetite (Fe₃O₄)coated or embedded in material that prevents the crystals frominteracting. Examples of such materials include proteins, such asferritin, polysaccharides, such as dextrans, or synthetic polymers suchas sulfonated polystyrene cross-linked with divinylbenzene. The coreportion is generally too small to hold a permanent magnetic field. Theferrofluids become magnetized when placed in a magnetic field. Examplesof ferrofluids and methods for preparing them are described by KemsheadJ. T. (1992) in J. Hematotherapy, 1:35-44, at pages 36 to 39, and Zioloet al. Science (1994) 257:219 which are incorporated herein byreference. Colloidal particles of dextran-iron complex are preferablyused in the process of the invention. (See Molday, R. S. and McKenzie,L. L. FEBS Lett. 170:232, 1984; Miltenyi et al., Cytometry 11:231, 1990;and Molday, R. S. and MacKenzie, D., J. Immunol. Methods 52:353, 1982;Thomas et al., J. Hematother. 2:297 (1993); and U.S. Pat. No. 4,452,733,which are each incorporated herein by reference).

FIG. 1 is a schematic representation of magnetic cell labeling usingtetrameric antibody complexes and colloidal dextran iron.

In accordance with the magnetic separation method, the sample containingthe progenitor and stem cells to be recovered, is reacted with the abovedescribed antibody reagents, preferably tetrameric antibody complexes,so that the antibody reagents bind to the targeted differentiated cellsand/or tumor cells present in the sample to form cell conjugates of thetargeted differentiated cells and/or tumor cells and the antibodyreagents. The reaction conditions are selected to provide the desiredlevel of binding of the targeted differentiated cells and/or tumor cellsand the antibody reagents. Preferably the sample is incubated with theantibody reagents for a period of 5 to 60 minutes at either 4° orambient room temperature. The concentration of the antibody reagents isselected depending on the estimated concentration of the targeteddifferentiated cells in the sample. Generally, the concentration isbetween about 0.1 to 50 μg/ml of sample. The magnetic particles are thenadded and the mixture is incubated for a period of about 5 minutes to 30minutes at the selected temperature. The sample is then ready to beseparated over a magnetic filter device. Preferably, the magneticseparation procedure is carried out using the magnetic filter andmethods described in U.S. Pat. No. 5,514,340 to Lansdorp and Thomaswhich is incorporated in its entirety herein by reference.

The sample containing the magnetically labelled cell conjugates ispassed through the magnetic filter in the presence of a magnetic field.In a preferred embodiment of the invention, the magnet is a dipolemagnet with a gap varying from 0.3 to 3.0 inches bore and having amagnetic field of 0.5-2 Tesla. The magnetically labelled cell conjugatesare retained in the high gradient magnetic column and the materialswhich are not magnetically labelled flow through the column afterwashing with a buffer.

The preparation containing non-magnetically labelled cells may beanalyzed using procedures such as flow cytometry. The ability of thecells in the preparation to produce colony-forming cells or long termculture initiating cells (LTCIC) in culture or repopulate SCID mice in aSCID repopulating assay (SRC) may also be assessed. The efficiency ofthe separation procedure may also be determined by monitoring therecovery of CD34⁺ cells, CD34⁺CD38⁻ cells and colony forming cells.

The antibody compositions of the invention may also be used to prepare acell preparation which is enriched for a specific differentiated celltype. This is achieved by using antibody compositions which lackantibodies to the specific differentiated cell type, in the abovedescribed processes of the invention. Particular embodiments of theseprocesses of the invention are set out below. It will be appreciatedthat the markers, matrices, antibody reagents, and procedures describedherein may be used in these processes to facilitate recovery of cellpreparations enriched for a specific differentiated cell type. Examplesof antibodies which may be used in these processes are set out in Table3.

In accordance with one embodiment of the invention, a process isprovided for enriching and recovering monocytes from a blood samplecomprising reacting the sample with an antibody composition containingantibodies capable of binding to the antigens glycophorin A, CD2, CD3,CD56, and CD24 or CD19, and optionally CD66b, under conditions so thatcell conjugates are formed between the antibodies and the cells in thesample having the antigens glycophorin A, CD2, CD3, CD56, and CD24 orCD19 and optionally CD66b on their surfaces; removing the cellconjugates; and recovering a cell preparation which is enriched inmonocytes.

In accordance with another embodiment of the invention, a process isprovided for enriching and recovering monocytes from a bone marrowsample comprising reacting the sample with an antibody compositioncontaining antibodies capable of binding to the antigens glycophorin A,CD2, CD3, CD56, and CD24 or CD19, and optionally CD66b and/or CD66e,under conditions so that cell conjugates are formed between theantibodies and the cells in the sample having the antigens glycophorinA, CD2, CD3, CD56, and CD24 or CD19 and optionally CD66b and/or CD66e ontheir surfaces; removing the cell conjugates; and recovering a cellpreparation which is enriched in monocytes.

In accordance with another embodiment of the invention, a process isprovided for enriching and recovering B-cells from a blood samplecomprising reacting the sample with an antibody composition containingantibodies capable of binding to the antigens glycophorin A, CD2, CD3,CD56, CD16 and CD14, and optionally CD66b under conditions so that cellconjugates are formed between the antibodies and the cells in the samplehaving the antigens glycophorin A, CD2, CD3, CD56, CD16 and CD14 andoptionally CD66b on their surfaces; removing the cell conjugates; andrecovering a cell preparation which is enriched in B-cells.

In accordance with another embodiment of the invention, a process isprovided for enriching and recovering B-cells from a bone marrow samplecomprising reacting the sample with an antibody composition containingantibodies capable of binding to the antigens glycophorin A, CD2, CD3,CD56, CD16 and CD14, and optionally CD66b and/or CD66e under conditionsso that cell conjugates are formed between the antibodies and the cellsin the sample having the antigens glycophorin A, CD2, CD3, CD56, CD16and CD14 and optionally CD66b and/or CD66e on their surfaces; removingthe cell conjugates; and recovering a cell preparation which is enrichedin B-cells.

In accordance with another embodiment of the invention, a process isprovided for enriching and recovering T-cells from a blood samplecomprising reacting the sample with an antibody composition containingantibodies capable of binding to the antigens glycophorin A, CD16, CD14,CD19, CD56, and optionally CD66b under conditions so that cellconjugates are formed between the antibodies and the cells in the samplehaving the antigens glycophorin A, CD16, CD14, CD19, CD56, andoptionally CD66b on their surfaces; removing the cell conjugates; andrecovering a cell preparation which is enriched in T-cells.

In accordance with another embodiment of the invention, a process isprovided for enriching and recovering T-cells from a bone marrow samplecomprising reacting the sample with an antibody composition containingantibodies capable of binding to the antigens glycophorin A, CD16, CD14,CD19, CD56, and optionally CD66b and/or CD66e under conditions so thatcell conjugates are formed between the antibodies and the cells in thesample having the antigens glycophorin A, CD16, CD14, CD19, CD56, andoptionally CD66b and/or CD66e on their surfaces; removing the cellconjugates; and recovering a cell preparation which is enriched inT-cells.

In accordance with yet another embodiment of the invention, a process isprovided for enriching and recovering CD4⁺T-cells from a blood samplecomprising reacting the sample with an antibody composition containingantibodies capable of binding to the antigens glycophorin A, CD16, CD14CD19, CD56, CD8, and optionally CD66b, under conditions so that cellconjugates are formed between the antibodies and the cells in the samplehaving the antigens glycophorin A, CD16, CD14, CD19, CD56, CD8, andoptionally CD66b on their surfaces; removing the cell conjugates; andrecovering a cell preparation which is enriched in CD4⁺T-cells.

In accordance with yet another embodiment of the invention, a process isprovided for enriching and recovering CD4⁺T-cells from a bone marrowsample comprising reacting the sample with an antibody compositioncontaining antibodies capable of binding to the antigens glycophorin A,CD16, CD14 CD19, CD56, CD8, and optionally CD66b and/or CD66e, underconditions so that cell conjugates are formed between the antibodies andthe cells in the sample having the antigens glycophorin A, CD16, CD14,CD19, CD56, CD8, and optionally CD66b and/or CD66e on their surfaces;removing the cell conjugates; and recovering a cell preparation which isenriched in CD4⁺T-cells.

In accordance with a further embodiment of the invention, a process isprovided for enriching and recovering CD8⁺T-cells from a blood samplecomprising reacting the sample with an antibody composition containingantibodies capable of binding to the antigens glycophorin A, CD16, CD14,CD19, CD56, CD4, and optionally CD66b under conditions so that cellconjugates are formed between the antibodies and the cells in the samplehaving the antigens glycophorin A, CD16, CD14, CD19, CD56, CD4, andoptionally CD66b on their surfaces; removing the cell conjugates; andrecovering a cell preparation which is enriched in CD8⁺T-cells.

In accordance with a further embodiment of the invention, a process isprovided for enriching and recovering CD8⁺T-cells from a bone marrowsample comprising reacting the sample with an antibody compositioncontaining antibodies capable of binding to the antigens glycophorin A,CD16, CD14, CD19, CD56, CD4, and optionally CD66b and/or CD66e underconditions so that cell conjugates are formed between the antibodies andthe cells in the sample having the antigens glycophorin A, CD16, CD14,CD19, CD56, CD4, and optionally CD66b and/or CD66e on their surfaces;removing the cell conjugates; and recovering a cell preparation which isenriched in CD8⁺T-cells.

In accordance with a still further embodiment of the invention, aprocess is provided for enriching and recovering NK-cells from a bloodsample comprising reacting the sample with an antibody compositioncontaining antibodies capable of binding to the antigens glycophorin A,CD4, CD14, CD19, and CD3, and optionally CD66b under conditions so thatcell conjugates are formed between the antibodies and the cells in thesample having the antigens glycophorin A, CD4 CD14, CD19, and CD3, andoptionally CD66b on their surfaces; removing the cell conjugates; andrecovering a cell preparation which is enriched in NK-cells.

In accordance with a still further embodiment of the invention, aprocess is provided for enriching and recovering NK-cells from a bonemarrow sample comprising reacting the sample with an antibodycomposition containing antibodies capable of binding to the antigensglycophorin A, CD4 CD14, CD19, and CD3, and optionally CD66b and/orCD66e under conditions so that cell conjugates are formed between theantibodies and the cells in the sample having the antigens glycophorinA, CD4, CD14, CD19, and CD3, and optionally CD66b and/or CD66e on theirsurfaces; removing the cell conjugates; and recovering a cellpreparation which is enriched in NK-cells.

In accordance with another embodiment, a process is provided forenriching and recovering basophils from whole blood sample comprisingreacting the sample with an antibody composition containing antibodiescapable of binding to the antigens glycophorin A, CD2, CD3, CD14, CD15,CD16, CD19, CD24, CD34, CD36, CD56 and CD45RA under conditions so thatcell conjugates are formed between the antibodies and the cells in thesample having the antigens glycophorin A, CD2, CD3, CD14, CD15, CD16,CD19, CD24, CD34, CD36, CD56 and CD45RA on their surfaces; removing thecell conjugates; and recovering a cell preparation which is enriched inbasophils.

In accordance with a further embodiment, a process is provided forenriching and recovering dendritic cells from a blood sample comprisingreacting the sample with an antibody composition containing antibodiescapable of binding to the antigens glycophorin A, CD3, CD14, CD16, CD19,CD34, CD56 and CD66b under conditions so that cell conjugates are formedbetween the antibodies and the cells in the sample having the antigensglycophorin A, CD3, CD14, CD16, CD19, CD34, CD56 and CD66b on theirsurfaces; removing the cell conjugates; and recovering a cellpreparation which is enriched in dendritic cells. Dentritic can also begenerated by culturing cells enriched for progenitors or monocytes usingthe previously mentioned enrichment cocktails.

In accordance with yet a further embodiment, a process is provided forenriching and recovering granulocytes from whole blood sample comprisingreacting the sample with an antibody composition containing antibodiescapable of binding to the antigens glycophorin A, CD2, CD56, CD19, CD14and CD3 under conditions so that cell conjugates are formed between theantibodies and the cells in the sample having the antigens glycophorinA, CD2, CD56, CD19, CD14 and CD3 on their surfaces; removing the cellconjugates; and recovering a cell preparation which is enriched ingranulocytes.

IV. Uses of the Compositions and Processes of the Invention

The compositions and processes of the invention may be used in theprocessing of biological samples including blood in particular, cordblood and whole blood. It has also been found that the antibodycompositions of the invention can be used to prepare hematopoieticprogenitor and stem cell preparations from bone marrow samples,including previously frozen bone marrow samples.

The processes of the invention are preferably used to deplete or purgeerythrocytes, B and T lymphocytes, monocytes, NK cells, granulocytes,and/or tumor cells from samples to prepare hematopoietic progenitor andstem cell preparations for use in transplantation as well as othertherapeutic methods that are readily apparent to those of skill in theart. For example, bone marrow or blood can be harvested from a donor inthe case of an allogenic transplant and enriched for progenitor and stemcells by the processes described herein.

Using the process of the invention it is possible to recover a highlypurified preparation of human hematopoietic stem/progenitor cells. Inparticular, a hematopoietic cell population containing greater than 50%of the hematopoietic progenitor/stem cells present in the originalsample, and which is depleted of differentiated cells and/or tumor cellsin the original sample by greater than 3 logarithms may be obtained. Thehuman hematopoietic progenitor and stem cells in the preparation are notcoated with antibodies, or modified making them highly suitable fortransplantation and other therapeutic uses that are readily apparent tothose skilled in the art.

The processes and compositions of the invention permit the isolation andrecovery of mature dendritic cells and their precursors from blood(Horrocks et al., In press.). Dendritic cells have many usefulapplications including as antigen presenting cells capable of activatingT cells both in vitro and in vivo. As an example, dendritic cells can beloaded (pulsed) in vitro with a tumor antigen and injected in vivo toinduce an anti-tumor T cell response.

The cell preparations obtained using the processes of the invention maybe used to isolate and evaluate factors associated with thedifferentiation and maturation of human hematopoietic cells. The cellpreparations may also be used to determine the effect of a substance oncell growth and/or differentiation into a particular lineage

The antibody compositions and processes of the invention may also beused to prepare a cell preparation from samples such as blood and bonemarrow, which is enriched in a selected differentiated cell type. Thiswill enable studies of specific cell to cell interactions includinggrowth factor production and responses to growth factors. It will alsoallow molecular and biochemical analysis of specific cells types. Cellpreparations enriched in NK cells and T-cells may also be used in immunetherapy against certain malignancies.

The tumor-enriching antibody composition of the invention is adapted toenrich for tumor cells, in particular non-hematopoietic metastatic tumorcells. The composition is useful in the detection of non-hematopoietictumor cells from blood, bone marrow, and peritoneal and pleuraleffusions of patients to aid in the diagnosis and detection ofmetastatic disease, monitoring the progression of metastatic disease, ormonitoring the efficacy of a treatment. The tumor enriching antibodycomposition applied in one step to a sample of peripheral blood, frozenperipheral blood, or bone marrow containing tumor cells results at leasta 2 log enrichment (and typically 3-4 log) of the tumor cells.

One currently used method for enriching for non-hematopoietic tumorcells is to use a negative selection technique with antibodies specificfor CD45. The inventors have compared their antibody composition withanti-CD45 alone on the ability to enrich peripheral blood mononuclearcells for breast carcinoma tumor cells and have shown that the antibodycomposition of the invention enriches the tumor cells 10 fold (1 log)over anti-CD45 alone.

The present invention also includes a useful kit in preparing a cellpreparation enriched in hematopoietic stem cells and progenitor cellscomprising antibodies specific for glycophorin A, CD3, CD24, CD16, CD14and instructions for preparing a cell preparation enriched inhematopoietic stem cells and progenitor cells.

The present invention further includes a kit useful in preparing a cellpreparation enriched in hemotopoietic stem and progenitor cells anddepleted in hematopoietic tumor cells comprising antibodies specific forglycophorin A, CD3, CD24, CD16, CD14 and instructions for preparing acell preparation enriched in hematopoietic stem cells and progenitorcells and depleted in hematopoietic tumor cells.

The present invention also includes a kit useful in preparing a cellpreparation enriched in hemotopoietic stem cells and progenitor cellsand depleted in tumor cells comprising antibodies specific forglycophorin A, CD3, CD24, CD16, CD14, and an antigen present on thetumor cells and instructions for preparing a cell preparation enrichedin hematopoietic stem cells and progenitor cells and depleted in tumorcells.

The present invention also relates to kits useful in preparing apreparation of non-hematopoietic tumor cells comprising antibodiesspecific for glycophorin A, CD2, CD14, CD16, CD38, CD45 and CD66b andinstructions for performing the tumor cell enriching processes of theinvention.

The following non-limiting examples are illustrative of the presentinvention:

EXAMPLES Example 1

Method for Evaluating Antibody Combinations

Suspensions of normal human bone marrow, human cord blood, mobilizedhuman peripheral blood and previously frozen human bone marrow werelabelled with tetrameric antibodies and colloidal dextran iron formagnetic cell depletions. Monoclonal antibodies recognizing lineagespecific cell surface antigens were mixed with a mouse IgG₁ anti-dextranantibody (Thomas, T. E, et al. (1992), J. Immunol Methods 154:245;252)and a rat IgG₁ monoclonal antibody which recognizes the Fc portion ofthe mouse IgG₁ molecule (TFL-P9) (Lansdorp, P. M, and Thomas, T. E.(1990), Mol. Immunol. 27:659-666). Tetrameric antibody complexes(Lansdorp, P. M, and Thomas, T. E. (1990), Mol. Immunol. 27:659-666;U.S. Pat. No. 4,868,109 to Lansdorp) spontaneously form when mouse IgG₁molecules (the lineage specific monoclonal antibody and anti-dextran)are mixed with P9. A proportion of these tetrameric antibody complexesare bifunctional, recognizing an antigen on the surface of the targetcell on one side and dextran (part of the magnetic colloidal dextraniron) on the other. Tetrameric antibody complexes were made for all theantibodies in the lineage cocktail. FIG. 1 shows a schematicrepresentation of magnetic cell labeling using tetrameric antibodycomplexes and colloidal dextran iron.

Cells were labelled for separation (1-5×10⁷ cells/ml) by incubating themwith the desired combination of tetramers for 30 min on ice followed bya 30 min incubation with colloidal dextran iron (final OD450=0.6)(Molday and MacKenzie 1982, 52(3): 353-367). The cells were then passedthrough a magnetic filter (U.S. Pat. No. 5,514,340; inventors Lansdorpand Thomas) at 1 cm/min. The magnetically labelled cells bind to thefilter and the unlabeled cells pass through. FIG. 1 shows a schematicrepresentation of magnetic cell labeling using tetrameric antibodycomplexes and colloidal dextran iron.

The flow through fraction is collected and analyzed for hematopoieticcolony forming cells (CFU-GM, CFU-C, LTCIC) (Eaves, C. J. and Eaves, A.J. 1992 In: Current Therapy in Hematology-Oncology, Fourth Edition pp.159-167), CD34⁺ cells, and CD34⁺CD38⁻ cells. The enrichment of thesecell types depends on how well the antibody cocktail has targeted othercells for removal. Each antibody cocktail was evaluated for the purityand recovery of colony forming cells, CD34⁺ cells, and CD34⁺CD38⁻ cells.FIG. 2 shows a FACS histogram of mobilized peripheral blood before andafter progenitor enrichment via lineage depletion.

Example 2

Antibodies for the Enrichment of Progenitor Cells (Progenitor Cocktail)

The results of numerous cell separations identified a combination oflineage specific antibodies that produce the maximum enrichment andrecovery of CD34⁺ cells and colony forming cells. TargetingErythrocytes—Anti-glycophorin A antibodies were used to labelerythrocytes for depletion. Many of these antibodies will causeagglutination at moderate to high antibody concentrations (3-10 μg/ml).It was found that cells could be effectively targeted for magneticdepletion with concentrations of anti-glycophorin antibody that wereseveral fold lower than that which caused agglutination. TargetingLymphocytes—B, T, and NK cells were targeted with monoclonal antibodiesagainst CD24, CD3, CD19, CD20, CD56, CD2. Initial depletions ofmobilized peripheral blood using just anti-CD24 and CD3 for lymphocytedepletion showed that a proportion of the CD34 negative cells in thepurified fraction were CD56 positive (NK cells). Subsequent tests withand without anti-CD56 increased the purity of CD34⁺ cells in therecovered fraction by 12-20%. Adding an anti-CD2 to the cocktailincreased the purity an additional 12-13%. Anti-CD2 and anti-CD56 had nosignificant effect on lineage depletions of fresh bone marrow. It islikely that bone marrow does not have as many CD3⁻ CD2⁺ and CD3⁻ CD56⁺cells. Anti-CD2 and anti-CD56 are added to the depletion cocktails formobilized peripheral blood but not bone marrow.

Antibodies against CD24 were sufficient to target all detectable B-cellsfor depletion. Adding anti-CD19 gave no additional enrichment of CD34⁺cells from mobilized peripheral blood or bone marrow. Substituting CD19for CD24 in separations of fresh bone marrow had no effect on theenrichment or recovery of CD34⁺ cells, hematopoietic colony formingcells and LTCIC. Replacing anti-CD24 with anti-CD20 or both anti-CD19and anti-CD20 had no significant effect on separations of mobilizedperipheral blood. An effect was seen in a separation with cord blood;when anti-CD24 was replaced with anti-CD19, the purity was decreased21%, but recovery of CD34⁺ cells was increased 28%. Targeting MatureMyeloid Cells—Monocytes were effectively targeted with an antibodyagainst CD14 in all cell suspensions tested. The removal of granulocytesfrom peripheral blood and fresh bone marrow was more efficient usingboth anti-CD16 and anti-CD66b rather than anti-CD16 alone and addinganti-CD66e gave an additional 10% enrichment of CD34⁺ cells from freshbone marrow and 20% enrichment for peripheral blood. Anti-CD16 alone wassufficient to deplete the granulocytes from previously frozen marrow.Adding anti-CD41 or CD42a did not increase the purity of CD34⁺ cells ineither peripheral blood or bone marrow.

Example 3

Antibodies for the Enrichment of Stem Cells (Stem Cell Cocktail)

Both pluripotent stem cells and committed progenitors express CD34, butthe CD34 compartment can be further subdivided using a variety of cellsurface markers to isolate these cell types. Stem cells co-purify in apopulation of CD34⁺ cells which lack or have low expression of certainlineage markers (CD38, CD33, CD45RA, CD71, CD36 and HLA-DR) (Craig etal. 1994, British Journal of Haematology, 88:24-30; Lansdorp, P. AI. andDragowska, W. 1992, J. Exp. Med. 175:1501-1509; Sutherland, H. J., etal. 1989, Blood 74.1563-1570.). If antibodies recognizing these antigensare included in the lineage cocktail one can further enrich for stemcells while losing some of the committed mature CD34⁺ cells. Antibodiesto CD36, CD38 and CD45RA were added to the lineage cocktail tospecifically enrich for stem cells. The recovery of CD34⁺CD38⁻ cells(Tables 5) and LTCICs (Table 6) were monitored to determine theefficiency of the lineage depletions with the “stem cell cocktail”.

Including anti-CD45RA in the stem cell cocktail does not negate the needfor anti-CD24 in the cocktail nor does anti-CD36 allow the removal ofCD66e. Adding anti-CD36 did increase the purity of CD34⁺CD38⁻ cells inseparations of previously frozen bone marrow by 15%. The addition ofanti-transferrin (CD71) antibody to the cocktail resulted in very poorrecovery of CD34⁺CD38⁻ cells, and LTCIC as well as producing asignificant number of dead cells in the enriched fraction (viabilitynormally >95%).

The results shown in Tables 5 and 6 demonstrate that separation of bloodand bone marrow with the stem cell cocktail produced a cell suspensionwhich is at least 30% and up to about 80% CD34⁺CD38⁻ with up to 90%recovery of these cells. FIG. 3 shows a FACS profile of peripheral bloodbefore and after progenitor enrichment with the antibody cocktail.

Example 4

Different Antibodies To The Same Antigen

The enrichment of hematopoietic progenitor cells via lineage depletionis not only dependent on the number of types of committed cells that aretargeted but also the effectiveness of this targeting and subsequentremoval using magnetic separation or other antibody mediated techniques.It was found that different antibodies recognizing the same antigen mayreproducibly produce different degrees of progenitor enrichment. Theanti-CD24 antibody 32D12 produced better results in lineage depletionsthan ALB9 (also anti-CD24); the purity of the enriched cell suspensionincreased 10% in a separation with cord blood. In cell depletions with asingle tetramer type, 32D12 out performed ALB9 and anti-glycophorinantibody 10F7MN out performed anti-glycophorin antibody D2.10 althoughswitching anti-glycophorin antibodies in a lineage depletion had nosignificant effect.

The criteria for choosing a particular antibody at a given concentrationis its performance in a magnetic cell separation which equates to themaximum depletion of antibody targeted cells with the maximum recoveryof non-target cells. Often depletion of anybody targeted cells increaseswith antibody concentration but so does the non-specific labeling ofcells. In general, the result looked for was a 3 log depletion withgreater than 75% recovery of CD34⁺ cells or non-targeted lymphocytes ifthe test cell suspension was steady state peripheral blood. Theperformance in a cell separation typically mimicked the degree ofspecific cell labeling and the degree on non-specific labeling measuredby FACS (sheep anti-mouse FHTC staining). Staining experiments wereoften run to eliminate antibodies (specific staining low, non-specificstaining high) and reduce the number of antibody concentrations to betested in cell separations.

Example 5

Purging Breast Carcinoma Cells (BT20 Or T47D Cells)

Tetramers of anti-breast carcinoma antibodies as shown in Table 4 werecombined with a progenitor enrichment cocktail (D2.10, UCHT1, MEM15,3G8, ALB9, 80H3, J4.119, 6F10.3, T199, and optionally 8D2.2, T16 andFA60152, or 1OF7MN, UCHT1, 32D12, MEM154, MEM15 or B13.9, T199, 6F10.3,J4.119, and optionally, 8D2.2, T16 and 1VC7) to produce a cocktail forbreast carcinoma purging and debulking. Including the lineage depletionincreases the degree of tumor purge over that seen with just anti-tumorantibodies alone (Table 7). Breast carcinoma cell lines were added topreviously frozen marrow, peripheral blood leukapheresis or fresh bonemarrow. Tumor cell purges were performed using the anti-breast carcinomaantibodies indicated in Table 7 with and without the standard lineagedepletion (progenitor enrichment cocktail). The recovery ofhematopoietic progenitors during lineage depletion is given in Table 8.Enrichment of progenitors was generally 50 to 100 fold.

In summary, purging tumor cells for hematopoietic progenitors in a onestep selection using the antibody cocktail as indicated in Table 7achieves a much higher degree of tumor cell purging than positiveselection techniques while offering a similar degree of progenitorenrichment. The recoveries of hematopoietic progenitor cells in alineage depletion are greater than those typically seen with positiveselection.

Example 6

Enrichment Of Breast Carcinoma Cells in Bone Marrow

Cells from the CAMA breast carcinoma cell line were mixed withpreviously frozen bone marrow (BM) and processed with the enrichmentantibody composition (D2.10, UCHT1, MEM15, 3G8, 80H3, J4119, 6F10.3,T199, 8D2.2, T16, FA6.152, and J33) in a one step magnetic depletion.The results shown in Table 9 demonstrates that the CAMA cells wereenriched 2-3 log using the tumor enrichment antibody compositions.

Example 7

Enrichment Of Breast Carcinoma Cells in Peripheral Blood

Cells from the CAMA breast carcinoma cell line were seeded intopreviously frozen peripheral blood mononuclear cells (PBMC) andprocessed with the enrichment antibodies capable of binding toglycophorin A (2B7.1), CD2 (6710.3), CD14 (MEM15), CD16 (3G8), CD38(T16), CD45 (J33) and CD66b (80H3) in a one step magnetic depletion. Theresults shown in Table 10 demonstrate that CAMA cells were enriched upto 4.5 log.

Example 8

Comparison with Antibodies to CD45

Cells from the CAMA breast carcinoma cell line were seeded intopreviously frozen peripheral blood mononuclear cells (PBMC) andprocessed with the enrichment antibodies capable of binding toglycophorin A (2B7.1), CD2 (6710.3), CD14 (MEM15), CD16 (3G8), CD38(T16), CD45 (J33) and CD66b (80H3). The results were compared with thecommon method of negative selection, ie. the use of anti-CD-45 alone.The results shown in Table 11, demonstrates that there is close to a tenfold (1 log) greater enrichment using the antibody composition of theinvention, over negative selection with CD45.

Example 9

Enrichment of Epithelial Tumor Cells From Pleural Effusion Samples

Pleural effusion samples were taken from patients with suspectedmetastatic disease. The pleural effusions were separated using the tumorenrichment composition of antibodies capable of binding to glycophorin A(2B7.1), CD2 (6710.3), CD14 (MEM15), CD16 (3G8), CD38 (T16), CD45 (J33)and CD66b (80H3). As shown in Table 12, there is up to a 2.5 logenrichment using the antibody composition of the invention.

Example 10

Enrichment of Epithelial Tumors Cells From Pleural Effusion SamplesDiluted into Peripheral Blood

Pleural effusion samples were taken from patients with suspectedmetastatic disease and seeded into previously frozen PBMC to mimicmetastatic cells in the blood, and then separated using the tumorenrichment composition of antibodies capable of binding to glycophorin A(2B7.1), CD2 (6710.3), CD14 (MEM15), CD16 (3G8), CD38 (T16), CD45 (J33)and CD66b (80H3). As shown in Table 13, there is up to a enrichmentusing the antibody composition of the invention, with up to a 4.9 logenrichment and 95% recovery.

While what is shown and described herein constitutes various preferredembodiments of the subject invention, it will be understood that variouschanges can be made to such embodiments without departing from thesubject invention, the scope of which is defined in the appended claims.

TABLE 1 Optimal Antibody Cocktail for the Enrichment of HematopoieticProgenitors optimum antibody Cell Suspension cocktail anti- % purityCD34+ cells % recovery CD34+ cells fresh bone marrow gly*, CD3, CD24,39,44,38 67,48,55 CD16, CD14, CD66e, CD66b, previously frozen gly, CD3,CD24, CD16, 64,46,50,53 85,55,82,64 bone marrow CD14, mobilized gly,CD3, CD24, CD16, 51,50,57 43,49,85 peripheral blood CD14, CD66e, CD66b,CD56, CD2, CD19 cord blood gly, CD3, CD24, CD16, 56,88,58,55,6375,85,53,67,48 CD14, CD66e, CD66b, CD56, CD2, CD19 *gly = glycophorin A

TABLE 2 Antibodies used in Lineage Depletions Concentration AntigenAntibody Source ug/ml glycophorin 10F7MN* U.S. Pat. No. 4,752,582 1 AD2.10 IMMUNOTECH, Marseille, France 2 2B7.1 StemCell Technologies 1 CD26F10.3 IMMUNOTECH, Marseille, France 3 CD3 UCHT1 IMMUNOTECH, Marseille,France 3 SK7 Becton Dickinson Immunocytometry, Mountain View, Calif. CD413B8.2 Becton Dickinson Immunocytometry, Mountain View, Calif. 3 CD8B911 Becton Dickinson Immunocytometry, Mountain View, Calif. 3 OKT3BioDesigns 3 CD14 MEM 15 Dr. Vaclav Horejsi, Institute of Molecular 2MEM 18 Genetics Academy of Sciences of the Czech Republic, Praha, Czech2 Republic; Cedarlane Laboratories Hornby, Ontario, Canada CD16 MEM 154*Dr. Vaclav Horejsi, Institute of Molecular 2 Genetics Academy ofSciences of the Czech Republic, Praha, Czech Republic, CedarlaneLaboratories Hornby, Ontario, Canada 3G8 IMMUNOTECH, Marseille, France 3NKP15 Becton Dickinson Immunocytometry, Mountain View, Calif. 3 CD19J4.119 IMMUNOTECH, Marseille, France 3 4G7 Becton DickinsonImmuncytometry, Mountain View, Calif. CD20 MEM97 Dr. Vaclav Horejsi,Institute of Molecular 3 Genetics Academy of Sciences of the CzechRepublic, Praha, Czech Republic; Cedarlane Laboratories Hornby, Ontario,Canada L27 Becton Dickison Immunocytometry, Mountain View, Calif. 3 CD2432D12* Dr. Steinar Funderud, Institute for Cancer 2 Research, Dept ofImmunology, Oslo, Norway ALB9 IMMUNOTECH, Marseille, France 3 CD36FA60152 IMMUNOTECH, Marseille, France 3 IVC7 CLB, Central Laboratory ofthe Netherlands, Red Cross Blood Transfusion Service CD38 T16IMMUNOTECH, Marseille, France 3 CD41 PI1.64 Kaplan, 5th InternationalWorkshop on Human Leukocyte 3 Differentiation Antigens CD42a Bebl BectonDickinson Immunocytometry, Mountain View, Calif. 3 CD45 J33 IMMUNOTECH,Marseille, France 3 MEM28 Dr. Vaclav Horejsi, Institute of Molecular 1CD45RA 8D2.2 Craig et al. 1994, StemCell Technologies, Vancouver, Canada1 L48 Becton Dickinson Immunocytometry, Mountain View, Calif. 3 CD56T199 IMMUNOTECH, Marseille, France 3 MY31 Becton DickinsonImmunocytometry, Mountain View, Calif. 3 CD66e CLB/gran10 CLB, CentralLaboratory of the Netherlands, Red Cross Blood 3 Transfusion ServiceCD66b B13.9 CLB, Central Laboratory of the Netherlands, Red Cross Blood3 Transfusion Service 80H3 IMMUNOTECH, Marseille, France 3 *preferredantibody based on performance in magnetic cell separations

TABLE 3 Antibody Cocktails to Purify Specific Types of Lineage CommittedCells Desired Cell type source of cells cocktail of antibodies MonocytesFicolled Blood anti-glycophorin A, anti-CD2,CD3,CD56,CD19 Whole Bloodanti-glycophorin A, anti-CD2,CD3,CD56,CD19,C66b B-Cells Ficolled Bloodanti-glycophorin A, anti-CD3,CD56,CD14,CD16,CD2 Whole Bloodanti-glycophorin A, anti-CD3,CD56,CD14,CD66b,CD16,CD2 T-Cells FicolledBlood anti-glycophorin A, anti-CD19,CD56,CD16,CD14 Whole Bloodanti-glycophorin A, anti-CD19,CD56,CD66b,CD16,CD14 CD4+ T-Cells FicolledBlood anti-glycophorin A, anti-CD19,CD56,CD8,CD16,CD14 Whole Bloodanti-glycophorin A, anti-CD19,CD56,CD8,CD66b,CD16,CD14 CD8+ T-CellsFicolled Blood anti-glycophorin A, anti-CD19,CD56,CD4,CD16,CD14 WholeBlood anti-glycophorin A, anti-CD19,CD56,CD4,CD66b,CD16,CD14 NK CellsFicolled Blood anti-glycophorin A, anti-CD19,CD3,CD14,CD4 Whole Bloodanti-glycophorin A, anti-CD19,CD3,CD66b,CD14,CD4 Basophils Whole Bloodanti-glycophorin A, anti-CD2, anti-CD3, anti-CD14, anti-CD15, anti-CD16,anti-CD19, anti-CD24, anti-CD34, anti-CD36, anti-CD56 and anti-C45RADendritic Cells Whole Blood anti-glycophorin A, anti-CD3, anti-CD14,anti-CD16, anti-CD19, anti-CD34, ant-CD56 and anti-CD66b Granulocyteswhole Blood anti-glycophorin A, anti-CD2, anti-CD56, anti-CD19,anti-CD14, and anti-CD3

TABLE 4 Antibodies Recognizing Non-Hematopoietic Antigens Expressed onEpithelial Tumor Cells. Disease Antibody Antigen Supplier/DeveloperBreast and Lung 5E11 unknown, breast carcinoma STI Carcinoma 6E7unknown, breast carcinoma STI H23A unknown, breast carcinoma ATCCRAR9941 epithelial glycoprotein Baxter, Germany RAR9948 epithelialglycoprotein Baxter, Germany RAR9938 crb2 Baxter, Germany C13B5 crb2Immunotech, Marseille, France BRST 1 BCA 225 ID Labs BRST 3 TAG-72 IDLabs CA15.3 MAM-6, mucin ID Labs CA27.29 MAM-6, mucin Cedarlane BerEp4HEA DAKO Neuroblastoma UJ13A unknown Hurko and Walsh (1983) Neurology33:734 UJ181.4 unknown Hurko and Walsh (1983) Neurology 33:734 UJ223.8unknown Hurko and Walsh (1983) Neurology 33:734 UJ127.11 unknown Hurkoand Walsh (1983) Neurology 33:734 5.1.H11 unknown Hurko and Walsh (1983)Neurology 33:734 390,459 unknown R.C. Seeger, L.A. Children's Hospital,Calif. BA-1.2 unknown R.C. Seeger, L.A. Children's Hospital, Calif. HSAN1.2 unknown Reynolds and Smith (1982) Hybridomas in Cancer p235

TABLE 5 Purity and Yield of Human CD34+ CD38− Cells Obtained Using thePrimitive Progenitor Enrichment Procedure % CD34+ CD38− % CD34+ CD38− %Yield Cell Sample n Start Fraction Enriched Fraction CD34+ CD38− CellsMobilized PB 3 0.02 ± 0.01 67 ± 6 50 ± 5  Frozen CB 1 0.16 78 20 BM 30.03 ± 0.01  61 ± 11 80 ± 10 Frozen BM 6 0.05 ± 0.01 34 ± 6 90 ± 20 %CD34+ CD38− cells in start fraction are typically too low to detectaccurately, therefore values given are rough estimates. Accordingly, %recovery values, which represent the present ratio of input vs.recovered absolute numbers of % CD34+ CD38− cells, are also relativelyinaccurate.

TABLE 6 Enrichment and Yield of Long-Term Culture Initiating Cells(LTC-IC) Using the Primitive Progenitor Enrichment Procedure Type ofCells % Yield of LTC-IC Fold-Enrichment of LTC-IC Mobilized PB 160 2,000 70 1,000 Frozen CB 180 4,000 BM 110 10,000  110 5,500

TABLE 7 Purging Breast Carcinoma Cells (BT20 or T47D cells). Anti-BreastCarcinoma Log Tumor Cell Cell Type Lineage Depletion AntibodiesDepletion Previously Frozen Bone Purge Only 5E11 1.8 Marrow 5E11, H23A3.7, 3.7 5E11, 6E7 3.0 Previously Frozen Bone Lineage Depletion andPurge 5E11 >5.8, 3.9, 4.7 Marrow RAR >5.8, 4.3, 4.7 BRST1 4.9 5E11,H23A >5.2, 4.4 5E11, RAR, BRST1 >5.8 Peripheral Blood Purge only 5E111.9, 1.9 Leukapheresis H23A 1.7 5E11, H23A 2.3 Peripheral Blood LineageDepletion and Purge 5E11, H23A 5.6 Leukapheresis Fresh Bone MarrowLineage Depletion and Purge 5E11, H23A 4.6, 4.4

TABLE 8 Recovery of Hematopoietic Colony Forming Cells During LineageDepletion To Enrich For Progenitors % Recovery Colony Assay mean rangeCFU-GM 60-100 75 BFU-E 71-100 92 LTCIC 72->100 100 

TABLE 9 Enrichment of CAMA Breast Carcinoma Tumor Cells From Bone Marrow#CAMA in % CAMA in % CAMA in % Recovery Log Enrich. Exp # Sample StartStart Flow CAMA CAMA 1 BM 1.1/10² 1.06 91.07 72.41 1.9 2 BM 2.2/10² 2.1896.40 44.12 1.6 2.1/10³ 0.21 82.16 75.00 2.6 2.1/10⁴ 0.02 32.01 60.003.2 3 BM 2.6/10³ 0.26 62.54 * 2.4 2.6/10⁴ 0.026 11.21 * 2.6 2.6/10⁵0.0026 2.01 * 2.9 2.6/10⁶ 0.00026 0.13 * 2.7 *Cell numbers were too lowto count accurately.

TABLE 10 Purity, Recovery, and Enrichment of CAMA Breast Carcinoma TumorCells Seeded into Previously Frozen Peripheral Blood Mononuclear CellsStart Enriched Fraction % Purity % Purity % Recovery Log Enrichment 0.395.5 9.0 2.5 0.03 65.3 13.3 3.4 0.003 17.1 12.1 3.8 0.3 96.7 14.3 2.50.03 52.4 13.1 3.3 0.003 82.7 14.8 4.5 0.3 92.7 25.8 2.5 0.03 61.4 17.03.3 0.003 16.8 7.0 3.7 0.3 91.2 14.5 2.5 0.03 61.3 17.9 3.3 0.003 18.08.0 3.8 0.02 24.5 47.2 3.1 0.02  9.3 42.9 2.7 0.1 97.7 85.8 2.9 0.0181.0 86.1 3.9 0.001 21.4 62.1 4.3 0.004  6.6 4.4 3.2 0.03 42.8 12.5 3.20.02 35.7 12.8 3.3 0.01 40.4 62.3 3.5 0.01 36.3 54.2 3.4 0.01 33.7 53.03.4 0.02 43.3 25.2 3.4 0.02 52.9 38.1 3.5 0.02 26.9 113.0 3.2 0.02 34.771.9 3.3

TABLE 11 Purity and Ennchment of CAMA Breast Carcinoma Tumor CellsSeeded into Previously Frozen Peripheral Blood Mononuclear Cells:Antibody Composition of the Invention vs. CD45 Depletion Only StartEnriched Fraction Cocktail % Purity % Purity Log Enrichment AntibodyCocktail 0.001 21.4 4.3 Antibody-CD45 only 0.001 6.2 3.8 AntibodyCocktail 0.03 42.8 3.2 Antibody-CD45 only 0.03 6.6 2.4 Antibody Cocktail0.02 35.7 3.3 Antibody-CD45 only 0.02 5.4 2.4 Antibody Cocktail 0.0140.4 3.5 Antibody-CD45 only 0.01 11.8 3.0 Antibody Cocktail 0.01 36.33.4 Antibody Cocktail 0.01 33.7 3.4 Antibody-CD45 only 0.01 8.0 2.8Antibody Cocktail 0.02 43.3 3.4 Antibody-CD45 only 0.02 20.1 3.1Antibody Cocktail 0.02 26.9 3.2 Antibody Cocktail 0.02 34.7 3.3Antibody-CD45 only 0.02 3.5 2.3

TABLE 12 Purity, Recovery, and Enrichment of Cytokeratin⁺ Cells fromPleural Effusions Sample Start Enriched Fraction # % Purity % Purity %Recovery Log Enrichment 1 0.3 85.9 22.5 2.5 2 1.5 84.0 10.0 1.8 3 4.456.2 24.8 1.1 4 2.9 94.4 14.9 1.5 5 2.8 87.7 21.2 1.5 6 19.5 35.4 67.50.3 7 17.4 99.6 11.7 0.8 8 0.3 57.6 6.9 2.2 9 2.1 93.4 9.2 1.6 10 0.316.7 0.5 1.8 11 3.7 91.8 7.6 1.4 12 2.3 86.5 61.7 1.6 13 0.0 3.6 14 12.382.2 14.8 0.8 15 74.6 94.5 8.6 0.1

TABLE 13 Purity, Recovery, and Enrichment of Cytokeratin⁺ Cells fromPleural Effusions Seeded into Previously Frozen Peripheral BloodMononuclear Cells Start Enriched Fraction Frequency % Purity % Purity %Recovery Log Enrichment 3/10⁵ 0.003 16.3 44.6 3.8 3/10⁶ 0.0003 2.4 42.33.9 9/10⁶ 0.0009 12.6 28.0 4.2 9/10⁷ 0.00009 0.8  8.6 3.9 1/10⁵ 0.00110.2 19.4 4.0 1/10⁶ 0.0001 1.4 31.6 4.2 2/10⁵ 0.0002 1.7 44.7 4.0 2/10⁷0.00002 1.1 95.1 4.8 2/10⁷ 0.00002 1.3 31.3 4.9

We claim:
 1. A process for enriching and recovering dendritic cells froma human sample comprising reacting the sample with an antibodycomposition containing antibodies capable of binding to the antigensglycophorin A, CD3, CD14, CD16, CD19, CD34, CD56 and CD66b underconditions so that conjugates are formed between the antibodies and thecells in the sample having the antigens glycophorin A, CD3, CD14, CD16,CD19, CD34, CD56 and CD66b on their surfaces; removing the conjugates;and recovering a cell preparation which is enriched in dendritic cells.2. A process according to claim 1, wherein the sample is whole blood. 3.A process according to claim 1, wherein the antibodies in the antibodycomposition are monoclonal antibodies.
 4. A process according to claim1, wherein the antibodies in the antibody composition are labelled witha marker or they are conjugated to a matrix.
 5. A process according toclaim 1, wherein the antibodies in the antibody composition are labelledwith biotin or a fluorochrome.
 6. A process according to claim 4,wherein the matrix is magnetic beads, a panning surface, dense particlesfor density centrifugation, an adsorption column, or an adsorptionmembrane.
 7. A process according to claim 3, wherein each of themonoclonal antibodies in the antibody composition is incorporated in atetrameric antibody complex which comprises a first monoclonal antibodyof a first animal species from the antibody composition, and a secondmonoclonal antibody of the first animal species which is capable ofbinding to at least one antigen on the surface of a matrix, which havebeen conjugated to form a cyclic tetramer with two monoclonal antibodiesof a second animal species directed against the Fc-fragments of theantibodies of the first animal species.